Process for producing partial glyceride

ABSTRACT

The invention relates to a process for preparing a partial glyceride, which includes, in a glycerolysis reaction of oil or fat making use of a lipase, conducting the reaction in the presence of water under conditions that crystals are partially precipitated in the reaction system in the course of the reaction and the concentration of free fatty acids in an oil phase amounts to at least 5% by weight.

This application is a 371 of PCT/JP99/03632 filed Jul. 6, 1997.

TECHNICAL FIELD

The present invention relates to a preparation process of partialglycerides making good use of a glycerolysis reaction of oil or fat by alipase.

BACKGROUND ART

Preparation processes of partial glycerides include an esterificationprocess and a glycerolysis process, and each process includes casesmaking use of a chemical catalyst and cases making use of an enzymecatalyst. As an esterification process making use of an enzyme catalyst,there has been known a process in which partial glycerides are preparedfrom oleic acid or the like high in fatty acid purity and glycerol asdescribed in Japanese Patent Publication No. 56311/1994 and JapanesePatent Application Laid-Open No. 19090/1987. However, this process isnot preferable as the preparation process of the partial glycerides fromthe economical point of view because a fatty acid prepared bydecomposing oil or fat at a high temperature and a high pressure andthen removing a colored component by a distilling treatment is used, andglycerol obtained by dehydration, concentration of purificationtreatments of an about 10% aqueous solution of glycerol produced uponthe decomposition of the oil or fat is used. Accordingly, theglycerolysis process in which both fatty acid group and glycerol groupconstituting the partial glycerides are derived from a cheap oil or fat,and a deficient glycerol group can be supplied as glycerol is effectiveas the industrial production process of the partial glycerides.

The processes for preparing the partial glycerides from such a cheap oilor fat and glycerol by an enzymatic glycerolysis reaction are reportedin Japanese Patent Publication No. 12710/1992, JAOCS, 71, 3, 339 (1994),etc.

However, the process described in Japanese Patent Publication No.12710/1992 requires a special lipase satisfying conditions under whichno secondary production of free fatty acids in a low water contentregion is attendant, so that it is difficult to preset a water contentin a reaction system and procure the lipase. On the other hand, in theprocess described in JAOCS, 71, 3, 339 (1994), the partial glyceridesare prepared from hydrogenated hardened beef tallow and glycerol by aglycerolysis process by which the formation of fatty acids is inhibited.However, this process has involved a problem that reproducibility of thereaction is poor.

Accordingly, it is an object of the present invention to provide aprocess for preparing a partial glyceride with industrial advantage byan enzymatic glycerolysis reaction.

DISCLOSURE OF THE INVENTION

The present inventors have found that when in a glycerolysis reaction ofoil or fat making use of an easily available lipase, the reaction isconducted in a system in which water is present, under conditions thatthe secondary production of fatty acids occurs to a great extent andthat crystals are precipitated in the reaction system, which areentirely different from the usual common sense, the yield of a partialglyceride, particularly a diglyceride is enhanced with great strides,thus leading to completion of the present invention.

According to the present invention, there is thus provided a process forpreparing a partial glyceride, which comprises, in a glycerolysisreaction of oil or fat making use of a lipase, conducting the reactionin the presence of water under conditions that crystals are partiallyprecipitated in the reaction system in the course of the reaction andthe concentration of free fatty acids in an oil phase amounts to atleast 5% by weight.

According to the present invention, there is also provided a process forpreparing a diglyceride, which comprises conducting a removing operationof fatty acid, glycerol and monoglyceride components from a reactionmixture in the reaction described above or after completion of thereaction.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, oil or fat, a lipase, glycerol and water areused as raw materials. Examples of the oil or fat include vegetable oilsand animal oils, more specifically, soybean oil, rapeseed oil,cotton-seed oil, corn oil, rice oil and fish oil. Oil or fat, in which aproportion of saturated fatty acids in the acyl group is at most 30% byweight, is preferably used. When the content of the saturated fattyacids is high, not only a high temperature is required of the reaction,but also a reaction product cannot be taken out of a vessel after thereaction, or heating is required, and so handling is complicated becausethe melting point of the raw oil or fat itself is high and the meltingpoint of a partial glyceride formed is higher. In addition, a problem ofdeactivation of the enzyme arises in the high-temperature and high-watercontent system.

No particular limitation is imposed on the lipase used. However, anenzyme (1,3-position-selective lipase”) specifically acting on the 1-and 3-positions of glycerol is preferred. More preferably, lipasesderived from microorganisms of the genera Rhizopus, Aspergillus andMucor, and splenic lipases, more specifically, lipases derived fromRhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Aspergillusniger, Mucor javanicus and Mucor miehei may be used. Immobilized lipasesobtained by immobilizing each of the lipases on various kinds ofcarriers (Celite, diatomaceous earth, silica gel, ion-exchange resin,etc.) may also be used. In general, the use of the latter lipases, i.e.,(heat-resistant) immobilized lipases having high durability in a wideconcentration range is preferred to the use of the former lipases. Manyof these lipases or immobilized lipases are easily available as productson the market. The amount of the lipase used is preferably 0.1 to 30% byweight, particularly 1 to 15% by weight (200 to 100,000 units per g ofoil or fat) based on the raw materials for the reaction.

No particular limitation is imposed on the water content in the processaccording to the present invention. However, it is preferably 5 to 50%by weight, particularly 8 to 30% by weight, in terms of an initialconcentration in the reaction system, based on glycerol from theviewpoint of acceleration of the reaction. With the progress of thereaction, the water content in the reaction system somewhat varies.However, the reaction is scarcely affected thereby.

Fatty acids and partial glycerides may exist in the raw materials forthe reaction in addition to the oil or fat, glycerol and water. However,the number of moles of glycerol group is preferably controlled to arange of 0.3 to 3, particularly 0.8 to 1.5 per mole of the fatty acidgroup in the whole raw material.

The reaction conditions in the present invention require that crystalsare partially precipitated in the reaction system in the course of thereaction and the concentration of free fatty acids in an oil phaseamounts to at least 5% by weight. The conditions under which crystalsare partially precipitated in the reaction system in the course of thereaction are conditions that oil or fat to be reacted is naturallymelted in the initial stage of the reaction, but parts of monoglyceridesand free fatty acids formed with the progress of the reaction areprecipitated.

Thus, it is assumed that both of monoglycerides and free acids arepartly excluded from the phase system of the reaction when brought inthe condition under which a part of crystals is precipitated in thecourse of the reaction, whereby the glycerolysis reaction is furtheraccelerated. Such conditions vary according to the oil or fat used as araw material. When oil or fat, in which a proportion of saturated fattyacids in the acyl group is at most 30% by weight, is used as the oil orfat, the conditions are preferably achieved by presetting the reactiontemperature to a range of 0 to 250° C., particularly 0 to 150° C.

In the present invention, the conditions are preset in such a mannerthat the concentration of free fatty acids in an oil phase amounts to atleast 5% by weight, preferably 8 to 30% by weight, the yield of thepartial glycerides is enhanced with great strides. Such conditions arepreferably achieved by presetting the reaction temperature to a range of0 to 25° C., particularly 0 to 15° C. like the conditions for theprecipitation of crystals.

The fact that the yield of the partial glycerides is enhanced by thepresetting of these reaction conditions is contrary to the idea of theconventional processes described in Japanese Patent Publication No.12710/1992 and the like that conditions that the water content is lowand no free fatty acid is formed are preset. This is considered to beattributable to the fact that the conventional view of glycerolysisreaction mechanism have been wrong. More specifically, the glycerolysisreaction of oil or fat (TG) with glycerol have generally been consideredto be as follows. Namely, a fatty acid group in the oil or fat is bondedto glycerol (GLY) to form monoglycerides (MGs) and diglycerides (DGs) asshown in the following scheme. The water content in the reaction systemat this time is considered to be a factor that the enzymatic activity(reaction rate) is changed. Namely, the reaction has been considered tobe an ester exchange reaction or alcoholic group exchange reaction inwhich no fatty acid is formed.

However, when an experiment of the glycerolysis reaction is conducted,hydrolysis occurs even in a region of a trace water content to formfatty acids. Even when the rate equations of the respective componentsare calculated out from the above formula to conduct kinetic analysis,the presence of the fatty acids formed cannot be theoretically proven.The reaction of a lipase has generally been considered to take place atan interface between an oil phase and a water phase. However, thiscannot be explained since the reaction occurs even in a nonaqueoussystem containing no water.

Therefore, the reaction mechanism of the glycerolysis in the case wherea 1,3-position-selective lipase is used as an enzyme catalyst isconsidered to be as follows. Namely, no alcohol group (ester) exchangereaction takes place, and hydrolysis, ester synthesis, conversion intoMG and conversion into DG only take place. A reaction site is consideredto be a contact point between the oil phase and the enzyme, and theconcentrations of the respective components in the rate equation areregarded as concentrations in the oil phase. More specifically, it hasbeen considered that the concentrations of the fatty acids andglycerides constituting the oil phase, and the concentration ofdissolved water and GLY contribute to the reaction, while water and GLYpotion in the aqueous solution phase of glycerol do not contribute tothe reaction and only affect the concentrations of water and GLY in theoil phase. When experimental data were subjected to fitting (numericalanalysis) using simultaneous differential equations of the rate equationon such hypothesis, the calculated result consisted with theexperimental date with considerable accuracy. When the experimentalresults at various temperature in a range in which no crystal wasprecipitated were also subjected to numerical analysis to calculate outrespective rate constants. These values were Arrhenius-plotted againstthe temperatures. As a result, linearity is obtained. Therefore, thevalidity of the model was able to be proven, and so it is consideredthat the glycerolysis reaction progresses in accordance with thefollowing scheme.

More specifically, the mechanism that partial glycerides is formed fromoil or fat and glycerol using an enzyme as a catalyst is considered tobe as follows. Namely, TG is hydrolyzed to form DGs and fatty acids, thefatty acids and GLY are subjected to ester synthesis to form MGs, andfurther esterification occurs to synthesize DGs. Such elucidation of themechanism of the glycerolysis permits proving that the yield of thepartial glycerides is enhanced.

As described above, the reaction temperature is preferably suitablyadjusted according to the kind of the resin used at a temperature rangeof 0 to 25° C. (particularly 0 to 15° C.), and the reaction time issuitably 10 to 200 hours, particularly 20 to 100 hours.

The reaction may be further continued after removing the crystalsprecipitated in the course of the reaction outside the system. However,the crystals may be removed after completion of the reaction. In orderto collect the partial glycerides from the reaction mixture, it ispreferable to conduct the above crystal-removing operation, water phase-(GLY phase-) removing operation and distilling operation in suitablecombination.

In order to obtain diglycerides in the process according to the presentinvention, it is only necessary to conduct the removing operation of thefatty acid, glycerol and monoglyceride components from a reactionmixture. Parts of the fatty acid and monoglyceride components can beremoved by the crystal-removing operation. Water, glycerol and lipasecan be removed by the water phase-removing operation. The fatty acids,monoglycerides and glycerol in the oil phase can be efficiently removedby distillation, particularly molecular distillation. Accordingly, theseoperations are combined, whereby the intended product (diglycerideconcentration: at least 55% by weight, triglyceride concentration: atmost 30% by weight) high in diglyceride content can be obtained.

EXAMPLES

The present invention will hereinafter be described by the followingExamples.

Examples 1 to 5 and Comparative Examples 1 to 6

A 4-necked flask was charged with raw oil or fat (100 g), lipase powder(3 g; or 30 g in the case of an immobilized lipase), water (0 to 3 g)and glycerol (21 g; water content in glycerol: 0.150%) to conduct areaction for 72 hours with stirring at 450 rpm in a thermostatic chambercontrolled at 5 to 45° C. (Table 1). The contents were in the form of aliquid-liquid emulsion of an oil phase and an aqueous solution ofglycerol up to about 2 hours from the beginning of the reaction. InExamples and Comparative Examples, 1, 2 and 6, however, white crystalswere gradually precipitated after that, and a slurry state, in which thecrystals were present, was held until the reaction was completed.

After completion of the reaction, the reaction mixture containing thecrystals was heated to 70° C. to melt the crystals, and centrifugation(3,000 rpm, 5 minutes) was then conducted. As a result, the reactionmixture was separated into two phases. An upper layer separated is anoil phase (A) in which a triglyceride (TG), diglycerides (DGs),monoglycerides (MGs), fatty acids (FAs), soluble glycerol (GLY) andsoluble water were present, and a lower layer was an aqueous solution ofglycerol containing the enzyme. In the analysis of the oil phase, aconcentration of the fatty acids was calculated out from an acid valueand an average molecular weight, and concentrations of TG, DG, MG andGLY by trimethylsilylating them and then correcting each peak areaobtained by using gas chromatography from the peak area of a standardsample of its corresponding pure product. The water content wasdetermined by the Karl Fischer's method. The results are shown in Table2.

After completion of the reaction, the reaction mixture was subjected toultracentrifugation (40,000 rpm, for 1 hour) at the same temperature asthe reaction temperature. As a result, the reaction mixture wasseparated into an upper layer which was a transparent oil phase (B)containing no crystal and a lower layer which was a mixture of theenzyme, a crystal portion and an aqueous solution of glycerol. Asanalyses of the oil phase (B), in addition to the same analysis as inthe oil phase (A), compositional analysis of fatty acids (alkyl) wasconducted by hydrolyzing the oil phase (B), then conducting methylesterification and using gas chromatography, thereby determining theamount of saturated fatty acids. The results are shown in Table 3.

Further, the oil phase (B) was subjected to a molecular distillationtreatment under conditions of 240° C. and 6.45 Pa to concentrate DG intothe residual portion, thereby analyzing the compositions of glycerideand alkyl. The results are shown in Table 4.

Further, the residual portion is charged into a plastic container (PETbottle), and the plastic container was transferred to a thermostaticchambers controlled at 5° C. and 15° C., respectively, thereby observingthe degree of precipitation of crystals. The results are shown in Table5.

In the reactions in all of Examples, and Comparative Examples 1, 2 and6, crystals were precipitated in the course of the respective reactions.

In Comparative Examples 3, 4 and 5, no crystal was precipitated, and soultracentrifugation was not conducted.

TABLE 1 Amount of Amount of Enzyme Amount of Raw oil or fat oil or fatglycerol Water species enzyme Temp. Ex. 1 Soybean oil 100 g 21 g   3 gPL  3 g  5° C. Ex. 2 Soybean oil 100 g 21 g   3 g IM 30 g  5° C. Ex. 3Soybean oil 100 g 21 g   3 g PS  3 g  5° C. Ex. 4 Rapeseed oil 100 g 21g   3 g PL  3 g  5° C. Ex. 5 Lard 100 g 21 g   3 g PS  3 g 45° C. Comp.Ex. 1 Soybean oil 100 g 21 g   0 g IM 30 g  5° C. Comp. Ex. 2 Soybeanoil 100 g 21 g 0.3 g PL  3 g  5° C. Comp. Ex. 3 Soybean oil 100 g 21 g  3 g PL  3 g 40° C. Comp. Ex. 4 Soybean oil 100 g 21 g   3 g IM 30 g40° C. Comp. Ex. 5 Rapeseed oil 100 g 21 g   3 g PL  3 g 40° C. Comp.Ex. 6 Beef Tallow 100 g 21 g 0.8 g PS  3 g 45° C. Water content in theraw glycerol: 0.15% PL: Lipase PL (Alcaligenes sp.; product of MeitoSangyo Co., Ltd.) IM: Lipozyme IM (Mucor miehei, product of Novo NordiskBioindustory Co.) PS: Lipase PS (Psedomonas fluorescens; product ofAmano Pharmaceutical Co., Ltd.)

Water content in the raw glycerol: 0.15%

PL: Lipase PL (Alcaligenes sp.; product of Meito Sangyo Co., Ltd.)

IM: Lipozyme IM (Mucor miehei, product of Novo Nordisk Bioindustory Co.)

PS: Lipase PS (Psedomonas fluorescens; product of Amano PharmaceuticalCo., Ltd.)

TABLE 2 Composition (% by weight) of Oil Phase (A) after completion ofthe reaction Fatty acid GLY MG DG TG DG/(DG + TG) × 100 Ex. 1 15.9 5.233.9 37.3  7.7 82.9 Ex. 2 17.9 2.0 24.5 43.2 12.6 77.4 Ex. 3 14.4 2.727.7 41.2 14.0 74.6 Ex. 4 18.4 7.0 27.8 35.7 11.1 76.3 Ex. 5 14.2 2.427.7 41.0 14.7 73.6 Comp. Ex. 1  1.3 0.1  1.9  9.9 86.8 10.2 Comp. Ex. 2 4.5 6.4 39.5 32.1 21.5 59.9 Comp. Ex. 3 14.1 1.6 13.0 41.5 29.8 58.2Comp. Ex. 4 12.9 0.7 13.3 38.6 34.5 52.8 Comp. Ex. 5 14.3 1.5 13.7 39.531.0 56.0 Comp. Ex. 6  4.7 5.5 25.1 39.9 24.8 61.7

TABLE 3 Composition of Oil Phase (B) after removing crystals CompositionFatty of alkyl, acid GLY MG DG TG C16 + C18 Ex. 1 19.5 3.3 26.9 41.0 9.3  4.6 Ex. 4 19.9 3.6 24.2 39.1 13.2  2.7 Ex. 5 16.8 1.8 22.1 43.715.6 30.9 Comp. Ex. 3* 14.1 1.6 13.0 41.5 29.8 14.8 Comp. Ex. 5* 14.31.5 13.7 39.5 31.0  5.3 Comp. Ex. 6  5.2 4.2 22.3 40.6 27.7 44.6 Thecomposition of the oil phase (A) in Comparative Examples because nocrystal was precipitated. C16 = Palmitic acid, C18 = Stearic acid.

The composition of the oil phase (A) in Comparative Examples because nocrystal was precipitated. C16=Palmitic acid, C18=Stearic acid.

TABLE 4 Composition of residue after molecular distillation treatmentComposition Fatty of alkyl, acid GLY MG DG TG C16 + C18 Ex. 1 0.1 0.01.2 74.8 23.9  3.3 Ex. 4 0.1 0.0 1.3 70.1 28.5  2.2 Ex. 5 0.2 0.0 1.466.7 31.7 27.8 Comp. Ex. 3 0.2 0.0 1.6 52.2 46.0 14.9 Comp. Ex. 5 0.10.0 1.1 51.4 47.4  5.2 Comp. Ex. 6 0.1 0.0 1.2 55.7 43.0 41.2

TABLE 5 Low-temperature preservation test of residue After 24 hrs. at15° C. After 24 hrs. at 5° C. Ex. 1 Transparent liquid Transparentliquid having flowability having flowability Ex. 2 Transparent liquidTransparent liquid having flowability having flowability Comp. Ex. 3Precipitate Having no flowability Sedimented; only due tocrystallization upper part had flowability as a whole, and not taken outof the bottle Comp. Ex. 5 Precipitate Having no flowability Sedimented;only due to crystallization upper part had flowability as a whole, andnot taken out of the bottle

From the above results, it was proven that in the preparation of thepartial glycerides by the glycerolysis reaction of oil or fat with alipase, (1) the yield is more enhanced when water is present in thereaction system, (2) the yield is more enhanced under conditions thatcrystals are precipitated in the course of the reaction and the contentof free fatty acids in an oil phase is high, and (3) the collection andpurification of the partial glycerides become difficult unless thereaction is conducted under high-temperature conditions when lard or thelike high in saturated fatty acid content is used, while handlingbecomes easy and purification can be easily conducted even underlow-temperature conditions when oil or fat low in saturated fatty acidcontent is used.

INDUSTRIAL APPLICABILITY

According to the present invention, high-purity partial glycerides canbe produced with industrial advantage under mild conditions.

What is claimed is:
 1. A process for preparing a partial glyceride,which comprises, in a glycerolysis reaction of oil or fat making use ofa lipase, conducting the reaction in the presence of water underconditions that crystals are partially precipitated in the reactionsystem in the course of the reaction and the concentration of free fattyacids in an oil phase amounts to at least 5% by weight.
 2. The processaccording to claim 1 for preparing a diglyceride, wherein the removingoperation of the fatty acid, glycerol and monoglyceride components is atleast one operation selected from (1) a removing operation of crystalsprecipitated in the reaction system in the reaction, (2) a removingoperation of a water phase and (3) a distilling operation.
 3. Theprocess according to claim 1 or claim 2 for preparing the partialglyceride, wherein oil or fat, in which a proportion of saturated fattyacids in the acyl group is at most 30% by weight, is used as the raw oilor fat.